Zhuren Wang

Modulation of Kv3 Subfamily Potassium Currents by the Sea Anemone Toxin BDS: Significance for CNS and Biophysical Studies

Kv3 potassium channels, with their ultra-rapid gating and high activation threshold, are essential for high-frequency firing in many CNS neurons. Significantly, the Kv3.4 subunit has been implicated in the major CNS disorders Parkinsons and Alzheimers diseases, and it is claimed that selectively targeting this subunit will have therapeutic utility. Previous work suggested that BDS toxins (“blood depressing substance,” from the sea anemone Anemonia sulcata) were specific blockers for rapidly inactivating Kv3.4 channels, and consequently these toxins are increasingly used as diagnostic agents for Kv3.4 subunits in central neurons. However, precisely how selective are these toxins for this important CNS protein? We show that BDS is not selective for Kv3.4 but markedly inhibits current through Kv3.1 and Kv3.2 channels. Inhibition comes about not by “pore block” but by striking modification of Kv3 gating kinetics and voltage dependence. Activation and inactivation kinetics are slowed by BDS-I and BDS-II, and V1/2 for activation is shifted to more positive voltages. Alanine substitution mutagenesis around the S3b and S4 segments of Kv3.2 reveals that BDS acts via voltage-sensing domains, and, consistent with this, ON gating currents from nonconducting Kv3.2 are markedly inhibited. The altered kinetics and gating properties, combined with lack of subunit selectivity with Kv3 subunits, seriously affects the usefulness of BDS toxins in CNS studies. Furthermore, our results do not easily fit with the voltage sensor “paddle” structure proposed recently for Kv channels. Our data will be informative for experiments designed to dissect out the roles of Kv3 subunits in CNS function and dysfunction.

The molecular basis of high-affinity binding of the antiarrhythmic compound vernakalant (RSD1235) to Kv1.5 channels.

Vernakalant (RSD1235) is an investigational drug recently shown to convert atrial fibrillation rapidly and safely in patients (J Am Coll Cardiol 44:2355–2361, 2004). Here, the molecular mechanisms of interaction of vernakalant with the inner pore of the Kv1.5 channel are compared with those of the class IC agent flecainide. Initial experiments showed that vernakalant blocks activated channels and vacates the inner vestibule as the channel closes, and thus mutations were made, targeting residues at the base of the selectivity filter and in S6, by drawing on studies of other Kv1.5-selective blocking agents. Block by vernakalant or flecainide of Kv1.5 wild type and mutants was assessed by whole-cell patch-clamp experiments in transiently transfected human embryonic kidney 293 cells. The mutational scan identified several highly conserved amino acids, Thr479, Thr480, Ile502, Val505, and Val508, as important residues for affecting block by both compounds. In general, mutations in S6 increased the IC50 for block by vernakalant; I502A caused an extremely local 25-fold decrease in potency. Specific changes in the voltage-dependence of block with I502A supported the crucial role of this position. A homology model of the pore region of Kv1.5 predicted that, of these residues, only Thr479, Thr480, Val505, and Val508 are potentially accessible for direct interaction, and that mutation at additional sites studied may therefore affect block through allosteric mechanisms. For some of the mutations, the direction of changes in IC50 were opposite for vernakalant and flecainide, highlighting differences in the forces that drive drug-channel interactions.

Regulation of transient Na+ conductance by intra- and extracellular K+ in the human delayed rectifier K+ channel Kv1.5

1 Significant Na+ conductance has been described in only a few native and cloned K+ channels, but has been used to characterize inactivation and K+ binding within the permeation pathway, and to refine models of K+ flux through multi‐ion pores. Here we use Na+ permeation of the delayed rectifier K+ channel Kv1.5 to study extra‐ and intracellular K+ (K+o and K+i, respectively) regulation of conductance and inactivation, using whole‐cell recording from human embryonic kidney (HEK)‐293 cells. 2 Kv1.5 Na+ currents in the absence of K+o and K+i were confirmed by: (i) resistance of outward Na+ currents to dialysis by K+‐free solutions; (ii) tail current reversal potential changes with Na+o with a slope of 55·8 mV per decade; (iii) block by 4‐aminopyridine (50 % at 50 μM), and resistance to Cl− channel inhibition. 3 Na+ currents were transient followed by a small sustained current. An envelope test confirmed that activated Kv1.5 channels conducted Na+, and that rapid current decay reflected C‐type inactivation. Sustained currents (≈13 % of peak) represented Na+ flux through inactivated Kv1.5 channels. 4 K+o could modulate the maximum available Na+ conductance in the stable cell line while channels were closed. Before the first pulse of a train, increasing K+o concentration increased the subsequent Na+ conductance from ≈15 (0 mM K+o) to 30 nS (5 mM K+o), with a Kd of 23 μM. Repeated low rate depolarizations in Na+i/Na+o solutions induced a use‐dependent loss of Kv1.5 channel Na+ conductance, distinct from that caused by C‐type inactivation. K+o binding that sensed little of the electric field could prevent this secondary loss of available Kv1.5 channels with a Kd of 230 μM. These two effects on conductance were both voltage independent, and had no effect on channel inactivation rate. 5 K+o concentrations ≥ 0·3 mM slowed the inactivation rate in a strongly voltage‐dependent manner. This suggested it could compete for binding at a K+ site or sites deeper in the pore, as well as restoring the Na+ conductance. K+i was able to modulate the inactivation rate but was unable to affect conductance. 6 Mutation of arginine 487 in the outer pore region of the channel to valine (R487V) greatly reduced C‐type inactivation in Na+ solutions, caused loss of channel use dependence, and prevented any conductance increase upon the addition of 0·1 mM K+o. Our results confirm the existence of a high affinity binding site at the selectivity filter that regulates inactivation, and also reveals the presence of at least one additional high affinity outer mouth site that predominantly regulates conductance of resting channels, and protects channels activated by depolarization when they conduct Na+.

A High-Na+ Conduction State during Recovery from Inactivation in the K+ Channel Kv1.5

Na(+) conductance through cloned K(+) channels has previously allowed characterization of inactivation and K(+) binding within the pore, and here we have used Na(+) permeation to study recovery from C-type inactivation in human Kv1.5 channels. Replacing K(+) in the solutions with Na(+) allows complete Kv1.5 inactivation and alters the recovery. The inactivated state is nonconducting for K(+) but has a Na(+) conductance of 13% of the open state. During recovery, inactivated channels progress to a higher Na(+) conductance state (R) in a voltage-dependent manner before deactivating to closed-inactivated states. Channels finally recover from inactivation in the closed configuration. In the R state channels can be reactivated and exhibit supernormal Na(+) currents with a slow biexponential inactivation. Results suggest two pathways for entry to the inactivated state and a pore conformation, perhaps with a higher Na(+) affinity than the open state. The rate of recovery from inactivation is modulated by Na(+)(o) such that 135 mM Na(+)(o) promotes the recovery to normal closed, rather than closed-inactivated states. A kinetic model of recovery that assumes a highly Na(+)-permeable state and deactivation to closed-inactivated and normal closed states at negative voltages can account for the results. Thus these data offer insight into how Kv1. 5 channels recover their resting conformation after inactivation and how ionic conditions can modify recovery rates and pathways.

Single-channel basis for the slow activation of the repolarizing cardiac potassium current, IKs

Coassembly of potassium voltage-gated channel, KQT-like subfamily, member 1 (KCNQ1) with potassium voltage-gated channel, Isk-related family, member 1 (KCNE1) the delayed rectifier potassium channel IKs. Its slow activation is critically important for membrane repolarization and for abbreviating the cardiac action potential, especially during sympathetic activation and at high heart rates. Mutations in either gene can cause long QT syndrome, which can lead to fatal arrhythmias. To understand better the elementary behavior of this slowly activating channel complex, we quantitatively analyzed direct measurements of single-channel IKs. Single-channel recordings from transiently transfected mouse ltk− cells confirm a channel that has long latency periods to opening (1.67 ± 0.073 s at +60 mV) but that flickers rapidly between multiple open and closed states in non-deactivating bursts at positive membrane potentials. Channel activity is cyclic with periods of high activity followed by quiescence, leading to an overall open probability of only ∼0.15 after 4 s under our recording conditions. The mean single-channel conductance was determined to be 3.2 pS, but unlike any other known wild-type human potassium channel, long-lived subconductance levels coupled to activation are a key feature of both the activation and deactivation time courses of the conducting channel complex. Up to five conducting levels ranging from 0.13 to 0.66 pA could be identified in single-channel recordings at 60 mV. Fast closings and overt subconductance behavior of the wild-type IKs channel required modification of existing Markov models to include these features of channel behavior.

Kif5b is an essential forward trafficking motor for the Kv1.5 cardiac potassium channel

We have investigated the role of the kinesin I isoform Kif5b in the trafficking of a cardiac voltage‐gated potassium channel, Kv1.5. In Kv1.5‐expressing HEK293 cells and H9c2 cardiomyoblasts, current densities were increased from control levels of 389 ± 50.0 and 317 ± 50.3 pA pF−1, respectively, to 614 ± 74.3 and 580 ± 90.9 pA pF−1 in cells overexpressing the Kif5b motor. Overexpression of the Kif5b motor increased Kv1.5 expression additively with several manipulations that reduce channel internalization, suggesting that it is involved in the delivery of the channel to the cell surface. In contrast, expression of a Kif5b dominant negative (Kif5bDN) construct increased Kv1.5 expression non‐additively with these manipulations. Thus, the dominant negative acts by indirectly inhibiting endocytosis. The increase in Kv1.5 currents induced by wild‐type Kif5b was dependent on Golgi function; a 6 h treatment with Brefeldin A reduced Kv1.5 currents to control levels in Kif5b‐overexpressing cells but had little effect on the increase associated with Kif5bDN expression. Finally, expression of the Kif5bDN prior to induction of Kv1.5 in a tetracycline inducible system blocked surface expression of the channel in both HEK293 cells and H9c2 cardiomyoblasts. Thus, Kif5b is essential to anterograde trafficking of a cardiac voltage‐gated potassium channel.

Shared requirement for dynein function and intact microtubule cytoskeleton for normal surface expression of cardiac potassium channels.

Potassium channels at the cardiomyocyte surface must eventually be internalized and degraded, and changes in cardiac potassium channel expression are known to occur during myocardial disease. It is not known which trafficking pathways are involved in the control of cardiac potassium channel surface expression, and it is not clear whether all cardiac potassium channels follow a common pathway or many pathways. In the present study we have surveyed the role of retrograde microtubule-dependent transport in modulating the surface expression of several cardiac potassium channels in ventricular myocytes and heterologous cells. The disruption of microtubule transport in rat ventricular myocytes with nocodazole resulted in significant changes in potassium currents. A-type currents were enhanced 1.6-fold at +90 mV, rising from control densities of 20.9 +/- 2.8 to 34.0 +/- 5.4 pA/pF in the nocodazole-treated cells, whereas inward rectifier currents were reduced by one-third, perhaps due to a higher nocodazole sensitivity of Kir channel forward trafficking. These changes in potassium currents were associated with a significant decrease in action potential duration. When expressed in heterologous human embryonic kidney (HEK-293) cells, surface expression of Kv4.2, known to substantially underlie A-type currents in rat myocytes, was increased by nocodazole, by the dynein inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride, and by p50 overexpression, which specifically interferes with dynein motor function. Peak current density was 360 +/- 61.0 pA/pF in control cells and 658 +/- 94.5 pA/pF in cells overexpressing p50. The expression levels of Kv2.1, Kv3.1, human ether-a-go-go-related gene, and Kir2.1 were similarly increased by p50 overexpression in this system. Thus the regulation of potassium channel expression involves a common dynein-dependent process operating similarly on the various channels.

Gating Charge Immobilization Caused by the Transition between Inactivated States in the Kv1.5 Channel

Sustained Na(+) or Li(+) conductance is a feature of the inactivated state in wild-type (WT) and nonconducting Shaker and Kv1.5 channels, and has been used here to investigate the cause of off-gating charge immobilization in WT and Kv1.5-W472F nonconducting mutant channels. Off-gating immobilization in response to brief pulses in cells perfused with NMG/NMG is the result of a more negative voltage dependence of charge recovery (V(1/2) is -96 mV) compared with on-gating charge movement (V(1/2) is -6.3 mV). This shift is known to be associated with slow inactivation in Shaker channels and the disparity is reduced by 40 mV, or approximately 50% in the presence of 135 mM Cs. Off-gating charge immobilization is voltage-dependent with a V(1/2) of -12 mV, and correlates well with the development of Na(+) conductance on repolarization through C-type inactivated channels (V(1/2) is -11 mV). As well, the time-dependent development of the inward Na(+) tail current and gating charge immobilization after depolarizing pulses of different durations has the same time constant (tau = 2.7 ms). These results indicate that in Kv1.5 channels the transition to a stable C-type inactivated state takes only 2-3 ms and results in strong charge immobilization in the absence of Group IA metal cations, or even in the presence of Na. Inclusion of low concentrations of Cs delays the appearance of Na(+) tail currents in WT channels, prevents transition to inactivated states in Kv1.5-W472F nonconducting mutant channels, and removes charge immobilization. Higher concentrations of Cs are able to modulate the deactivating transition in Kv1.5 channels and prevent the residual slowing of charge return.

Components of gating charge movement and S4 voltage-sensor exposure during activation of hERG channels.

The human ether-á-go-go–related gene (hERG) K+ channel encodes the pore-forming α subunit of the rapid delayed rectifier current, IKr, and has unique activation gating kinetics, in that the α subunit of the channel activates and deactivates very slowly, which focuses the role of IKr current to a critical period during action potential repolarization in the heart. Despite its physiological importance, fundamental mechanistic properties of hERG channel activation gating remain unclear, including how voltage-sensor movement rate limits pore opening. Here, we study this directly by recording voltage-sensor domain currents in mammalian cells for the first time and measuring the rates of voltage-sensor modification by [2-(trimethylammonium)ethyl] methanethiosulfonate chloride (MTSET). Gating currents recorded from hERG channels expressed in mammalian tsA201 cells using low resistance pipettes show two charge systems, defined as Q1 and Q2, with V1/2’s of −55.7 (equivalent charge, z = 1.60) and −54.2 mV (z = 1.30), respectively, with the Q2 charge system carrying approximately two thirds of the overall gating charge. The time constants for charge movement at 0 mV were 2.5 and 36.2 ms for Q1 and Q2, decreasing to 4.3 ms for Q2 at +60 mV, an order of magnitude faster than the time constants of ionic current appearance at these potentials. The voltage and time dependence of Q2 movement closely correlated with the rate of MTSET modification of I521C in the outermost region of the S4 segment, which had a V1/2 of −64 mV and time constants of 36 ± 8.5 ms and 11.6 ± 6.3 ms at 0 and +60 mV, respectively. Modeling of Q1 and Q2 charge systems showed that a minimal scheme of three transitions is sufficient to account for the experimental findings. These data point to activation steps further downstream of voltage-sensor movement that provide the major delays to pore opening in hERG channels.

NH2-terminal inactivation peptide binding to C-type-inactivated Kv channels.

In many voltage-gated K+ channels, N-type inactivation significantly accelerates the onset of C-type inactivation, but effects on recovery from inactivation are small or absent. We have exploited the Na+ permeability of C-type–inactivated K+ channels to characterize a strong interaction between the inactivation peptide of Kv1.4 and the C-type–inactivated state of Kv1.4 and Kv1.5. The presence of the Kv1.4 inactivation peptide results in a slower decay of the Na+ tail currents normally observed through C-type–inactivated channels, an effective blockade of the peak Na+ tail current, and also a delay of the peak tail current. These effects are mimicked by addition of quaternary ammonium ions to the pipette-filling solution. These observations support a common mechanism of action of the inactivation peptide and intracellular quaternary ammonium ions, and also demonstrate that the Kv channel inner vestibule is cytosolically exposed before and after the onset of C-type inactivation. We have also examined the process of N-type inactivation under conditions where C-type inactivation is removed, to compare the interaction of the inactivation peptide with open and C-type–inactivated channels. In C-type–deficient forms of Kv1.4 or Kv1.5 channels, the Kv1.4 inactivation ball behaves like an open channel blocker, and the resultant slowing of deactivation tail currents is considerably weaker than observed in C-type–inactivated channels. We present a kinetic model that duplicates the effects of the inactivation peptide on the slow Na+ tail of C-type–inactivated channels. Stable binding between the inactivation peptide and the C-type–inactivated state results in slower current decay, and a reduction of the Na+ tail current magnitude, due to slower transition of channels through the Na+-permeable states traversed during recovery from inactivation.